Phosphorylcholine conjugates and uses thereof

ABSTRACT

The present invention provides phosphorylcholine conjugates and pharmaceutical compositions comprising same for the prevention or treatment of autoimmune diseases. In particular, the conjugates of the present invention are effective in treating autoimmune diseases associated with pathological inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/956,167 filed on Dec. 1, 2015, which claims benefit to U.S.application Ser. No. 14/766,108 filed on Aug. 5, 2015, which is a 35U.S.C. § 371 National Phase Entry Application from InternationalApplication No. PCT/IL2014/050124 filed on Feb. 5, 2014, and designatingthe United States, which claims the benefit of U.S. ProvisionalApplication No. 61/760,668 filed on Feb. 5, 2013. The contents of all ofthese applications are hereby expressly incorporated by reference asfully set forth herein, in their entirety.

Field of the invention

The present invention relates to phosphorylcholine based agents andpharmaceutical compositions comprising same for the prevention andtreatment of autoimmune diseases, particularly autoimmune diseasesassociated with pathological inflammation.

BACKGROUND OF THE INVENTION

A strong correlation between improved sanitation and significantincrease in the prevalence of autoimmune and autoinflammatory syndromeshas been demonstrated in western countries. Moreover, a correlationbetween the presence of parasitic worms (helminths) in certaingeographic areas and protection from atopic, autoimmune, andautoinflammatory diseases was reported. These studies led to the“hygiene hypothesis”, postulating that the recent increase in autoimmunedisease incidences in the west reflects an absence of appropriatepriming of the immune response by infectious agents, including parasiticworms, during childhood.

During the last decades many studies reported that infection withparasitic helminthes, or systemic treatment with helminths extracts, canreduce inflammation associated with autoimmune diseases, such asmultiple sclerosis (MS), rheumatoid arthritis (RA), type I diabetesmellitus (T1DM), and inflammatory bowel disease (IBD).

Although such studies were successful, using potential pathogens astherapeutic agents has raised ethic and safety issues. Therefore,considerable effort has been spent in identifying and characterizing theparasite-derived molecules responsible for their immunomodulation.

The currently most-well defined nematode-derived immunomodulatorymolecule is ES-62. ES-62 is a tetrameric glycoprotein (62 kDa subunits)that has phosphorylcholine (PC)-moieties attached via an N-type glycan.

It has been proposed that the immunomodulatory activity of ES-62 isattributed to the presence of the PC moieties. Further support for thePC immunomodulatory activity was found in other parasitic nematodes likeAscaris suum that express only the PC-immunomodulatory moiety.

U.S. Pat. No. 5,455,032 discloses compositions useful for inducingimmunoprotection against infections by pathogenic organisms containingphosphocholine antigens, including Streptococcus pneumoniae and othermicroorganisms that have a phosphocholine antigen component on theirmembranes or capsids. Further disclosed are vaccines and methods forinducing immunoprotection against infection by these pathogenicorganisms.

U.S. Pat. No. 7,067,480 discloses the use of aphosphorylcholine-containing glycoprotein, particularly ES-62, in thetreatment or prophylaxis of autoimmune diseases associated with abnormalinflammation such as rheumatoid arthritis.

U.S. Pat. No. 8,012,483 discloses a method for identifying subjects atrisk of developing ischemic cardiovascular diseases by determining thepresence of antibodies, particularly IgM antibodies, towardphosphorylcholine and further discloses pharmaceutical compositionscomprising a phosphorylcholine conjugate, or an antibody withspecificity to a phosphorylcholine conjugate for active or passiveimmunogens in the treatment or prevention of atherosclerosis.

U.S. Patent Application, Publication No. 2010/0303721, discloses amethod of treating an excessive immune response including anaberrant/enhanced Th1 response with a helminthic parasite preparation.The autoimmune diseases includes Crohn's disease and ulcerative colitis,rheumatoid arthritis, type 1 diabetes mellitus, lupus erythematosus,sarcoidosis, multiple sclerosis, autoimmune thyroiditis, allergicrhinitis, colon polyps/colon cancer and asthma.

There remains a need to develop small molecules as safe and stableimmunomodulators, with minimal adverse side effects, for treatingautoimmune diseases, particularly disease associated with abnormalinflammation.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingphosphorylcholine (PC) conjugates and uses thereof for treatment and/orprevention of autoimmune diseases and disorders, such as, rheumatoidarthritis, lupus, multiple sclerosis, and inflammatory bowel disease.

The present invention is based in part on the unexpected discovery thatphosphorylcholine-conjugates comprising tuftsin derivatives (TD-PC),such as Thr-Lys-Pro-Arg-Gly-Tyr-PC (namely, SEQ ID NO: 25 conjugated toPC), exhibited an inhibitory and immunomodulatory effect on thedevelopment and progression of colitis, rheumatoid arthritis andsystemic lupus erythematosus (SLE) in vivo.

The present invention provides, in one aspect, aphosphorylcholine-conjugate comprising at least one phosphorylcholinederivative attached to at least one tuftsin derivative, the tuftsinderivative comprising a tuftsin attached to a plurality of amino acids,the plurality comprising a first amino acid attached to a second aminoacid, wherein the first amino acid is selected from the group consistingof glycine, alanine, leucine, valine, arginine, histidine, lysine,serine, threonine, asparagine, glutamine, isoleucine, methionine, andnon-natural, non-charged and non-polar amino acids, and wherein thesecond amino acid is selected from the group consisting of tyrosine andhistidine.

In certain embodiments, the first amino acid is selected from the groupconsisting of glycine, alanine, leucine, valine,β-alanine-6-aminohexanoic acid and 5-aminopentanoic acid. In certainembodiments, the first amino acid is selected from the group consistingof glycine, alanine, leucine and valine. Each possibility represents aseparate embodiment of the invention. In certain embodiments, the firstamino acid is glycine. Each possibility represents a separate embodimentof the invention.

In certain embodiments, the second amino acid is tyrosine. In certainembodiments, the second amino acid is tyrosine, and the PC derivative isp-aminophenylphosphorylcholine. In certain embodiments, the PCderivative is 4-aminophenylphosphorylcholine.

In certain embodiments, the second amino acid is tyrosine, the PCderivative is p-aminophenylphosphorylcholine, wherein said tyrosine isbound to the p-aminophenylphosphorylcholine

In certain embodiments, the second amino acid is histidine.

In certain embodiments, the phosphorylcholine-conjugate described abovecomprises one phosphorylcholine derivative attached to one tuftsinderivative. In certain embodiments, the phosphorylcholine-conjugatedescribed above comprises a plurality of phosphorylcholine derivativesattached to a plurality of tuftsin derivatives. In certain embodiments,the phosphorylcholine-conjugate described above comprises a plurality oftuftsin derivatives attached to one phosphorylcholine derivative. Incertain embodiments, the phosphorylcholine-conjugate described abovecomprises a plurality of phosphorylcholine derivatives attached to onetuftsin derivative.

In certain embodiments, the phosphorylcholine-conjugate described abovecomprises at least one phosphorylcholine derivative and the at least onetuftsin derivative are separated by a spacer.

In certain embodiments, the tuftsin derivative comprises an amino-acidsequence selected from the group consisting of SEQ ID NO: 23 to SEQ IDNO: 40. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the tuftsin derivative comprises the amino-acidsequence set forth in any one of SEQ ID NOs: 23 and 24.

In certain embodiments, the tuftsin derivative comprises the amino-acidsequence set forth in SEQ ID NO: 25.

In certain embodiments, the tuftsin derivative is consisting of theamino-acid sequence set forth in SEQ ID NO: 25.

The present invention further provides, in another aspect, apharmaceutical composition comprising any one of thephosphorylcholine-conjugates described above, and further comprises apharmaceutically acceptable carrier, diluent or excipient.

The present invention further provides, in another aspect, a method fortreating an autoimmune disease in a subject in need thereof comprisingadministering to the subject the pharmaceutical composition describedabove.

In certain embodiments, treating comprises at least one of preventingthe onset of said autoimmune disease, attenuating the progress of saidautoimmune disease and inhibiting the progression of said autoimmunedisease. In certain embodiments, treating comprises at least one ofattenuating the progress of said autoimmune disease and inhibiting theprogression of said autoimmune disease. Each possibility represents aseparate embodiment of the invention.

In certain embodiments, the subject is at high risk of developing saidautoimmune disease.

In certain embodiments, the autoimmune disease is associated withabnormal inflammation. In certain embodiments, the autoimmune disease isselected from the group consisting of rheumatoid arthritis, lupus,colitis, multiple sclerosis, pemphigus vulgaris, antiphospholipidsyndrome, psoriasis, autoimmune hepatitis, sarcoidosis, inflammatorybowel disease, crohn's disease and chronic obstructive pulmonarydisease. In certain embodiments, the autoimmune disease is selected fromthe group consisting of rheumatoid arthritis, lupus and colitis. Incertain embodiments, the autoimmune disease is lupus. In certainembodiments, the autoimmune disease is colitis. In certain embodiments,the autoimmune disease is rheumatoid arthritis. Each possibilityrepresents a separate embodiment of the invention.

In certain embodiments, the pharmaceutical composition is administeredin a route of administration selected from the group consisting of oral,intravenous, intramuscular, sublingual, intramucosal, intraperitoneal,nasal, subcutaneous, topical, and intradermal or transdermal. In certainembodiments, the pharmaceutical composition is orally administered. Eachpossibility represents a separate embodiment of the invention.

In certain embodiments, the subject is a mammal. In certain embodiments,the subject is human.

The present invention further provides, in another aspect, a kitcomprising a container comprising any one of thephosphorylcholine-conjugates described above.

In certain embodiments, the kit described above further comprises asecond container comprising a pharmaceutically acceptable carrier,diluent or excipient. In certain embodiments, the kit described abovefurther comprises instructions for preparing a pharmaceuticalcomposition from the contents of said first and said second containers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the level of anti-PC antibodies (in units of OpticalDensity, O.D., at 405 nm) in the sera of NZBxNZW/F1 mice (“lupus mice”)treated with PC-OVA (square; *=p<0.001), MPC (triangle; p>0.05) or PBS(control; diamond).

FIG. 2 shows the progression of proteinuria in lupus mice treated withPC-OVA (square), MPC (triangle) or PBS (control; diamond) as thepercentage of protein levels of at least 100 mg/dl (p<0.02) in theurine.

FIG. 3A shows kidney sections of lupus mice treated with PC-OVA probedwith anti-mouse IgG-FITC Fc specific antibodies.

FIG. 3B shows kidney sections of lupus mice treated with PC-OVA probedwith anti-mouse IgG-FITC Fc specific antibodies stained with Periodicacid—Schiff (PAS).

FIG. 4A shows kidney sections of lupus mice treated with MPC probed withanti-mouse IgG-FITC Fc specific antibodies.

FIG. 4B show kidney sections of lupus mice treated with MPC probed withanti-mouse IgG-FITC Fc specific antibodies stained with Periodicacid—Schiff (PAS).

FIG. 5A shows kidney sections of control lupus mice treated with PBSprobed with anti-mouse IgG-FITC Fc specific antibodies.

FIG. 5B and FIG. 5C show kidney sections of control lupus mice treatedwith PBS probed with anti-mouse IgG-FITC Fc specific antibodies stainedwith Periodic acid—Schiff (PAS).

FIG. 6 is a Kaplan-Meier graph demonstrating survival of lupus micetreated with PC-OVA (triangle; *=p<0.01), MPC (square; p<0.04) or PBS(control; diamond).

FIG. 7 shows the percentage of lupus mice with proteinuria (proteinlevel above 100 mg/dl) treated with PC-tuftsin (TPC) or PBS (control),p<0.02.

FIG. 8A shows PAS stained kidney sections of lupus mice treated with PBS(control).

FIG. 8B shows PAS stained kidney sections of lupus mice treated withPC-tuftsin.

FIG. 9A shows kidney sections of lupus mice treated with PBS (control)probed with anti-mouse IgG-FITC Fc specific antibodies.

FIG. 9B shows kidney sections of lupus mice treated with PC-tuftsinprobed with anti-mouse IgG-FITC Fc specific antibodies.

FIG. 10 shows relative mRNA expression levels of anti-inflammatorycytokine TGFβ (p<0.001) and pro-inflammatory cytokine IFNγ (p<0.03;relative to (β-actin) in the splenocytes of lupus mice treated withPC-tuftsin (TPC) or PBS.

FIG. 11 shows protein level (in units of pg/ml) of anti-inflammatorycytokines TGFβ and IL-10 and pro-inflammatory cytokines IFNγ and IL-17in the splenocytes of lupus mice treated with PC-tuftsin (TPC) or PBS(control). *=p<0.001, **=p<0.02.

FIG. 12 shows FACS analysis of T regulatory cells levels (Tregs—CD4⁺,CD25⁺, FOXP3⁺) in lupus mice treated with PC-tuftsin (TPC; p<0.02),phosphorylcholine (PC; p<0.01), tuftsin (T; p<0.01) or PBS (p<0.01).

FIG. 13 shows progression over time as a function of daily diseaseactivity (DAI; p<0.02) in mice subjected to DSS induction ofinflammatory bowel disease (“IBD mice”) following treatment withPC-tuftsin derivative (TD-PC) as set forth in SEQ ID NO: 25 (emptycircle) and PBS (solid circle).

FIG. 14 shows rectal bleeding in IBD mice following treatment withPC-tuftsin derivative as set forth in SEQ ID NO: 25 (empty circle) andPBS (solid circle) (p<0.001).

FIG. 15 shows weight loss in IBD mice following treatment withPC-tuftsin derivative as set forth in SEQ ID NO: 25 (TD-PC, emptycircle) and PBS (solid circle) relative to t=0 (p<0.001).

FIG. 16 shows survival of IBD mice following treatment with PC-tuftsinderivative (TD-PC) as set forth in SEQ ID NO: 25 (empty circle) and PBS(solid circle).

FIG. 17 shows colons of IBD mice following treatment with PC-tuftsinderivative (SEQ ID NO: 25; TD-PC) or PBS.

FIG. 18 shows colon length analysis (p<0.02) in IBD mice followingtreatment with PC-tuftsin derivative (TD-PC) or PBS.

FIG. 19A and FIG. 19D show H&E stained colon sections of IBD micetreated with PBS (control).

FIG. 19B and FIG. 19E show H&E stained colon sections of IBD micetreated with PC-tuftsin derivative.

FIG. 19C and FIG. 19F show H&E stained colon sections from a mouse notsubjected to DSS induction of IBD.

FIG. 20 shows immunoblot analysis of cytokines expression (TNFα, IL-17,IL-1β and IL-10) in the colons of mice following TD-PC or PBStreatments.

FIG. 21 shows the presence of proteinuria in NZBxW/F1 lupus control mice(PBS) or in mice treated with the TD-PC conjugate (TD-PC), the tuftsinderivative (TD) and phosphorylcholine (PC).

FIG. 22 is a Kaplan-Meier survival analysis of mice treated with TD-PC,tuftsin, PC or PBS.

FIG. 23 shows titers of circulating anti-dsDNA antibodies in the sera ofmice treated with TD-PC (TD-PC), the tuftsin derivative (TD),phosphorylcholine (PC) or PBS (V), obtained by ELISA at a dilution of1:800 (n=10 per group).

FIG. 24A shows the relative expression of IFNγ and TGFβ mRNAs in spleencells derived from TD-PC or PBS (control) treated mice.

FIG. 24B shows the level of pro-inflammatory cytokines (IFNγ, IL-17) andanti-inflammatory cytokines (IL-10, TGFβ) in the culture fluids ofsplenocytes originated from mice treated with TD-PC (TD-PC), the tuftsinderivative (TD), phosphorylcholine (PC) or PBS (V).

FIG. 25A shows FACS scans corresponding to the expansion of Tregs inmice treated with TD-PC (TD-PC), the tuftsin derivative (TD),phosphorylcholine (PC) or control (PBS).

FIG. 25B shows a quantitative analysis corresponding to the FACS scansrepresented in FIG. 25A.

FIG. 26A shows the arthritis score in mice following subcutaneous (s.c.)treatment with TD-PC or vehicle (PBS). *p<0.05, **p<0.01, ***p<0.001.

FIG. 26B shows the arthritis score in mice following treatment per os(oral) with TD-PC or vehicle (PBS). *p<0.05, **p<0.01, ***p<0.001.

FIG. 26C are representative photos of joints of control mice treatedwith vehicle (PBS) per os (panel A) or subcutaneously (panel B), and ofmice treated with TD-PC per os (panel C) or subcutaneously (panel D).

FIG. 27 shows the level of circulating anti-collagen antibodies in thesera of mice treated with TD-PC per os (oral), PBS per os (oral), TD-PCsubcutaneously (s.c.) and PBS subcutaneously (s.c.).

FIG. 28A shows the expression of the cytokine TNFα in the culture fluidsof splenocytes originated from mice treated with TD-PC or PBS, orally orsubcutaneously (s.c.).

FIG. 28B shows the expression of the cytokine IL-17 in the culturefluids of splenocytes originated from mice treated with TD-PC or PBS,orally or subcutaneously (s.c.).

FIG. 28C shows the expression of the cytokine IL-1β in the culturefluids of splenocytes originated from mice treated with TD-PC or PBS,orally or subcutaneously (s.c.).

FIG. 28D shows the expression of the cytokine IL-10 in the culturefluids of splenocytes originated from mice treated with TD-PC or PBS,orally or subcutaneously (s.c.).

FIG. 29A shows representative FACS scans associated with Treg cellsexpansion in isolated splenocytes derived from mice treated with TD-PCor control (PBS) mice.

FIG. 29B shows quantitative analysis corresponding to the FACS scansrepresented in FIG. 29A.

FIG. 30A and FIG. 30B show representative FACS scans associated withBreg cells expansion in isolated splenocytes derived from mice treatedwith TD-PC or control (PBS) mice.

FIG. 30C and FIG. 30D show analyses corresponding to FIG. 30A and FIG.30B, respectively.

FIG. 31A shows arthritis score in DBA/1 CIA control untreated mice (PBS)or mice treated with TD-PC conjugate (TD-PC), the tuftsin derivative(TD), phosphorylcholine (PC) and the tuftsin derivative combined withphosphorylcholine (TD+PC).

FIG. 31B are photos of representative joints of control CIA mice (PBS)and CIA mice treated with TD-PC (TD-PC), tuftsin, phosphorylcholine or amix of the tuftsin derivative with phosphorylcholine (TD+PC).

FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D and FIG. 32E show expressionlevels of the cytokines IL-1β; IL-17; IL-6; TNF-α; and IL-10,respectively, in the culture fluids of splenocytes originated fromuntreated mice, control mice (PBS) and mice treated with TD-PC, thetuftsin derivative (TD), phosphorylcholine (PC) or a mix of the tuftsinderivative with phosphorylcholine (TD+PC).

FIG. 33A shows representative FACS scans associated with Treg cellsexpansion in isolated splenocytes of derived from untreated mice (None),control mice (PBS) and mice treated with TD-PC (TD-PC), the tuftsinderivative (TD), phosphorylcholine (PC) and a mix of the tuftsinderivative with phosphorylcholine (TD+PC).

FIG. 33B shows an analysis corresponding to FIG. 33A.

FIG. 34A shows representative FACS scans associated with Breg cellsexpansion in isolated splenocytes derived from control mice (PBS) ormice treated with TD-PC, the tuftsin derivative (TD), phosphorylcholine(PC) and a mix of the tuftsin derivative with phosphorylcholine (TD+PC).

FIG. 34B shows quantitative analysis corresponding to FIG. 34A.

FIG. 35 shows relative secretion levels of anti-inflammatory IL-10cytokine from macrophages treated with TD-PC relative to PC-Ovalbumin(OVA-PC; p<0.00001).

FIG. 36A is a Principal Coordinate Analysis (PCA) representing bacteriapopulation in fecal samples of healthy mice or CIA mice prior to CIAinduction (day 0) and prior to treatment with TD-PC set forth in SEQ IDNO: 25 (gray square, gray triangle) or with a carrier (PBS; blacksquare, black circle).

FIG. 36B is a PCA representing bacteria population in fecal samples ofthe mice of FIG. 36B 35 days after initiating CIA in the relevant groups(squares), and after treating the mice with TD-PC set forth in SEQ IDNO: 25 (gray square and gray triangle) or with a carrier (PBS; blacksquare, black circle).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions comprising atleast one phosphorylcholine (PC) conjugated to at least one tuftsinderivative. According to some embodiments, the pharmaceuticalcompositions of the invention exhibit an immunomodulatory activity.According to some embodiments, the pharmaceutical compositions are fortreating, ameliorating the progress of, and preventing onset of,autoimmune diseases.

The present invention provides, in one aspect, aphosphorylcholine-conjugate comprising at least one phosphorylcholinederivative attached to at least one tuftsin derivative, the tuftsinderivative comprising a tuftsin (threonine-lysine-proline-arginine;TKPR; SEQ ID NO: 17) attached to a plurality of amino acids, theplurality comprising a first amino acid attached to a second amino acid,wherein the first amino acid is selected from the group consisting ofglycine, alanine, leucine, valine, arginine, histidine, lysine, serine,threonine, asparagine, glutamine, isoleucine, methionine, andnon-natural, non-charged and non-polar amino acids, and wherein thesecond amino acid is selected from the group consisting of tyrosine andhistidine.

The term “phosphorylcholine (PC) conjugate” as used herein, refers to aphosphorylcholine moiety or a derivative thereof linked to atuftsin-derivative (TD), optionally via a spacer.

The term “spacer”, as used herein, refers to a connecting or otherwisebridging element between the tuftsin derivative and the PC derivative,typically linked by chemical methods or biological means thereto.Non-limiting examples of spacers include: amino acids, peptides,polypeptides, proteins, hydrocarbons and polymers among others. Eachpossibility is a separate embodiment of the invention.

As used herein, the term “derivative of phosphorylcholine” includes, butis not limited to, the following: 4-amino-phenyl-phosphocholine,4-diazonio-phenyl-phosphorylcholine, 4-nitro-phenyl-phosphocholine and12-(3-iodophenyl)dodecyl-phosphocholine among others. Each possibilityis a separate embodiment of the invention.

As used herein, the term “tuftsin” refers to a tetrapeptide(threonine-lysine-proline-arginine, TKPR; SEQ ID NO: 17). Tuftsin may besynthesized chemically or isolated from the spleen by enzymatic cleavageof the Fc domain of IgG heavy chain. Tuftsin is known for itsphagocytosis-stimulating activity and augmentation of antigen presentingcapacity of macrophages in-vitro and in-vivo. According to someembodiments, tuftsin may be considered as an immunomodulatory molecule.

The terms “tuftsin derivative”, “TD” and “tuftsin-derived carriermoiety” are interchangeable and refer to tuftsin (THR-LYS-PRO-ARG; SEQID NO: 17) attached to at least two additional amino acids which areindependently selected. These terms thus include, but are not limitedto, the SEQ ID NOs presented in Table 1. Non-natural amino acids,preferably non-charged and non-polar non-natural amino acids such asβ-alanine-6-aminohexanoic acid and 5-aminopentanoic acid, may also becomprised in the tuftsin derivative.

The term “moiety” as used herein refers to a part of a molecule, whichlacks one or more atom(s) compared to the corresponding molecule. Theterm “moiety”, as used herein, further relates to a part of a moleculethat may include either whole functional groups or parts of functionalgroups as substructures. The term “moiety” further means part of amolecule that exhibits a particular set of chemical and/or pharmacologiccharacteristics which are similar to the corresponding molecule.

The term “attached to” as used herein refers to a covalent bond betweenat least two molecules or moieties. According to the principles of thepresent invention, the natural and non-natural amino-acids comprised inthe tuftsin derivative are adjacent and attached to one another, whilethe at least one phosphorylcholine derivative is attached to the atleast one tuftsin derivative either directly or indirectly via a spacer.

In certain embodiments, the first amino acid is selected from the groupconsisting of glycine, alanine, leucine, valine,β-alanine-6-aminohexanoic acid and 5-aminopentanoic acid. In certainembodiments, the first amino acid is selected from the group consistingof glycine, alanine, leucine and valine. Each possibility represents aseparate embodiment of the invention.

In certain embodiments, the first amino acid is glycine.

In certain embodiments, the second amino acid is tyrosine. In certainembodiments, the PC derivative is p-aminophenylphosphorylcholine. Incertain embodiments, the second amino acid is tyrosine and the PCderivative is p-aminophenylphosphorylcholine wherein the tyrosine isconjugated to said p-aminophenylphosphorylcholine. In certainembodiments, the second amino acid is histidine.

In certain embodiments, the phosphorylcholine-conjugate described abovecomprises one phosphorylcholine derivative attached to one tuftsinderivative. In certain embodiments, the phosphorylcholine-conjugatedescribed above comprises a plurality of phosphorylcholine derivativesattached to a plurality of tuftsin derivatives. In certain embodiments,the phosphorylcholine-conjugate described above comprises a plurality oftuftsin derivatives attached to one phosphorylcholine derivative. Incertain embodiments, the phosphorylcholine-conjugate described abovecomprises a plurality of phosphorylcholine derivatives attached to onetuftsin derivative.

In certain embodiments, the phosphorylcholine-conjugate described abovecomprises at least one phosphorylcholine derivative and the at least onetuftsin derivative are separated by a spacer.

TABLE 1 Amino-acid sequences of certain tuftsin derivatives SEQ IDAmino-acid sequences NO: Threonine-Lysine-Proline-Arginine-Xaa-Xaa 23Xaa-Xaa-Threonine-Lysine-Proline-Arginine 24Threonine-Lysine-Proline-Arginine-Glycine-Tyrosine 25Threonine-Lysine-Proline-Arginine-Glycine-Histidine 26Threonine-Lysine-Proline-Arginine-Alanine-Tyrosine 27Threonine-Lysine-Proline-Arginine-Alanine-Histidine 28Threonine-Lysine-Proline-Arginine-Leucine-Tyrosine 29Threonine-Lysine-Proline-Arginine-Leucine-Histidine 30Threonine-Lysine-Proline-Arginine-Valine-Tyrosine 31Threonine-Lysine-Proline-Arginine-Valine-Histidine 32Glycine-Tyrosine-Threonine-Lysine-Proline-Arginine 33Glycine-Histidine-Threonine-Lysine-Proline-Arginine 34Alanine-Tyrosine-Threonine-Lysine-Proline-Arginine 35Alanine-Histidine-Threonine-Lysine-Proline-Arginine 36Leucine-Tyrosine-Threonine-Lysine-Proline-Arginine 37Leucine-Histidine-Threonine-Lysine-Proline-Arginine 38Valine-Tyrosine-Threonine-Lysine-Proline-Arginine 39Valine-Histidine-Threonine-Lysine-Proline-Arginine 40

In certain embodiments, the tuftsin derivative comprises or consists ofan amino-acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO: 24. In certain embodiments, the tuftsin derivativecomprises or consists of an amino-acid sequence selected from the groupconsisting of SEQ ID NO: 25 to SEQ ID NO: 40. In certain embodiments,the tuftsin derivative comprises or consists of an amino-acid sequenceselected from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 32. Incertain embodiments, the tuftsin derivative comprises or consists of anamino-acid sequence selected from the group consisting of SEQ ID NO: 33to SEQ ID NO: 40. In certain embodiments, the tuftsin derivativecomprises or consists of an amino-acid sequence selected from the groupconsisting of SEQ ID NO: 25 to SEQ ID NO: 26. In certain embodiments,the tuftsin derivative comprises or consists of an amino-acid sequenceselected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29 and SEQ ID NO: 31. In certain embodiments, the tuftsinderivative comprises or consists of an amino-acid sequence selected fromSEQ ID NO: 25 and SEQ ID NO: 33. In certain embodiments, the tuftsinderivative comprises or consists of the amino-acid sequence of SEQ IDNO: 25. Each possibility represents a separate embodiment of theinvention.

The present invention further provides, in another aspect, apharmaceutical composition comprising any one of thephosphorylcholine-conjugates described above, and further comprises apharmaceutically acceptable carrier, diluent or excipient.

As used herein, the term “pharmaceutical composition” refers to anycomposition comprising at least one pharmaceutically active ingredientand at least one other ingredient, as well as any product which results,directly or indirectly, from combination, complexation, or aggregationof any two or more of the ingredients, from dissociation of one or moreof the ingredients, or from other types of reactions or interactions ofone or more of the ingredients. Accordingly, the term “pharmaceuticalcomposition” as used herein may encompass, inter alia, any compositionmade by admixing a pharmaceutically active ingredient and one or morepharmaceutically acceptable carriers.

The present invention further provides, in another aspect, a method fortreating an autoimmune disease in a subject in need thereof comprisingadministering to the subject the pharmaceutical composition describedabove.

As used herein, the terms “treating” or “treatment” are interchangeableand refer to any one or more of preventing the onset of an autoimmunedisease, attenuating the progress of said autoimmune disease andinhibiting the progression of an autoimmune disease, among others.

In certain embodiments, treating comprises at least one of preventingthe onset of said autoimmune disease, attenuating the progress of saidautoimmune disease and inhibiting the progression of said autoimmunedisease. In certain embodiments, treating comprises at least one ofattenuating the progress of said autoimmune disease and inhibiting theprogression of said autoimmune disease. Each possibility represents aseparate embodiment of the invention.

In certain embodiments, the subject is at high risk of developing saidautoimmune disease. As used herein, the term “high risk subject” is usedto mean a human who is considered to be at a higher risk of developingan autoimmune disease compared to the general population.

In certain embodiments, the autoimmune disease is associated withabnormal inflammation.

In certain embodiments, the autoimmune disease is selected from Acutedisseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, alopeciaareata, amyloidosis, ankylosing spondylitis, anti-GBM/Anti-TBMnephritis, antiphospholipid syndrome (APS), autoimmune angioedema,autoimmune aplastic anemia, autoimmune dysautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmuneoophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmunethrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmuneurticarial, axonal & neuronal neuropathies, Balo disease, Behcet'sdisease, bullous pemphigoid, cardiomyopathy, castleman disease, celiacdisease, chagas disease, chronic fatigue syndrome, chronic inflammatorydemyelinating polyneuropathy (CIDP), chronic recurrent multifocalostomyelitis (CRMO), Churg-Strauss syndrome, cicatricialpemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome,cold agglutinin disease, congenital heart block, coxsackie myocarditis,Crest disease, essential mixed cryoglobulinemia, demyelinatingneuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease(neuromyelitis optica), discoid lupus, Dressler's syndrome,endometriosis, eosinophilic esophagitis, eosinophilic fasciitis,erythema nodosum, experimental allergic encephalomyelitis, Evanssyndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis(temporal arteritis), giant cell myocarditis, Glomerulonephritis,Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerlycalled Wegener's Granulomatosis), Graves' disease, Guillain-Barresyndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolyticanemia, Henoch-Schonlein purpura, herpes gestationis,hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgAnephropathy, IgG4-related sclerosing disease, immunoregulatorylipoproteins, inclusion body myositis, interstitial cystitis, juvenilearthritis, juvenile diabetes (Type 1 diabetes), juvenile myositis,Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis,lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgAdisease (LAD), Lupus (SLE), lyme disease, chronic, Meniere's disease,microscopic polyangiitis, mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, Myastheniagravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's),neutropenia, Ocular cicatricial pemphigoid, optic neuritis, palindromicrheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus), paraneoplastic cerebellar degeneration,paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, pars planitis (peripheral uveitis),pemphigus, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatic,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, progesterone dermatitis, primary biliary cirrhosis, primarysclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathicpulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia,Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy,Reiter's syndrome, relapsing polychondritis, restless legs syndrome,retroperitoneal fibrosis, Rheumatic fever, rheumatoid arthritis,sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren'ssyndrome, sperm & testicular autoimmunity, stiff person syndrome,subacute bacterial endocarditis (SBE), Susac's syndrome, sympatheticophthalmia, Takayasu's arteritis, temporal arteritis/giant cellarteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome,transverse myelitis, type 1 diabetes, ulcerative colitis,undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis,vesiculobullous dermatosis, vitiligo and Wegener's granulomatosis (nowtermed granulomatosis with polyangiitis (GPA).

In certain embodiments, the autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, lupus, colitis, multiplesclerosis, pemphigus vulgaris, antiphospholipid syndrome, psoriasis,autoimmune hepatitis, sarcoidosis, inflammatory bowel disease, crohn'sdisease and chronic obstructive pulmonary disease. Each possibilityrepresents a separate embodiment of the invention.

In certain embodiments, the autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, lupus and colitis. In certainembodiments, the autoimmune disease is lupus. In certain embodiments,the autoimmune disease is colitis. In certain embodiments, theautoimmune disease is rheumatoid arthritis. Each possibility representsa separate embodiment of the invention.

In certain embodiments, the pharmaceutical composition is administeredin a route of administration selected from the group consisting of oral,intravenous, intramuscular, sublingual, intramucosal, intraperitoneal,nasal, subcutaneous, topical, intradermal and transdermal. In certainembodiments, the pharmaceutical composition is orally administered. Eachpossibility represents a separate embodiment of the invention.

In certain embodiments, the subject is a mammal. In certain embodiments,the subject is human.

The present invention further provides, in another aspect, a kitcomprising a container comprising any one of thephosphorylcholine-conjugates described above.

The term “kit” is typically meant a collection or array of certainequipment and chemicals (e.g. conjugates, buffers, etc.).

In certain embodiments, the kit described above further comprises asecond container comprising a pharmaceutically acceptable carrier,diluent or excipient. In certain embodiments, the kit described abovefurther comprises instructions for preparing a pharmaceuticalcomposition from the contents of said first and said second containers.

According to some embodiments, the TD-PC-conjugates of the presentinvention are produced synthetically to ensure the stability andreproducibility of these compounds, while maintaining or even enhancingtheir activity. According to some embodiments, TD-PC-conjugates areuseful as stimulators of a subject immune system towards the Th2phenotype.

As used herein, the terms “immunomodulation”, “immunomodulationactivity”, “immunomodulatory activity” or “modulating the immuneresponse” with reference to the TD-PC-conjugates of the presentinvention refer to the ability of the conjugates to elicit at least oneof: reducing the ability of lymphocytes (both B- and T-) to proliferatein response to antigen; inducing generation of T and/or B regulatory(suppressor) cells; and affecting macrophages and dendritic cellfunctions. Affecting macrophages and dendritic cell functions includesbut is not limited to, stimulating clearance of apoptotic cells andinducing tolerogenic dendritic cells; inhibiting the ability ofmacrophages to produce pro-inflammatory cytokines such as IL-12, TNF-αand IL-6; modulating dendritic cell maturation to preferentially elicitTh2-like responses; and inducing spleen cells to produce theanti-inflammatory cytokine, IL-10 and to bias antibody responses in aTh2/anti-inflammatory direction. The immunomodulatory activity of theTD-PC-conjugates may be also referred to herein as “anti-inflammatory”activity.

The tuftsin-derived (TD) carrier moiety may be immunogenic ornon-immunogenic.

As used herein, the term “immunogenic carrier” refers to a variety ofmolecules or substances that are capable of inducing an immune responseagainst the PC molecule.

In the Gram-positive bacterium Streptococcus pneumonia PC is attacheddirectly to sugar residues, generally considered to beN-acetylgalactosamine. PC has been detected also in a wide range ofGram-positive bacteria including Clostridium, Lactococcus, Bacillus andthe Gram-negative bacterium Haemophilus influenzae. Eukaryotic organismsin which PC has been detected include many important disease-causingagents such as the protozoa Leishmania major and Trypanosoma cruzi; awide range of fungi; the trematode Schistosoma mansoni; the tapewormDiphyllobothrium latum; several gastrointestinal nematodes and allspecies of filarial nematode. In human, PC appears on the inner leafletof a cell membrane and is exposed to the immune system by apoptoticcells.

The term “non-immunogenic carrier” as used herein refers to a variety ofmolecules or substances that do not elicit an immune response.

According to some embodiments, the TD-PC-conjugate comprises PC and atuftsin derivative, linked to one another.

As used herein, the term “linked” refers to attached, connected, boundto, in association with and coupled to, among others.

According to some embodiments, the PC and the tuftsin derivative arelinked through a covalent bond.

According to additional embodiments, the synthetic TD-PC-conjugates ofthe present invention may be synthesized as described in the Examplesection hereinbelow.

According to other embodiments, the pharmaceutical composition is in theform of solution, suspension, tablets, chewable tablets, capsules,syrups, intranasal sprays, suppositories, transdermal patches, amongother types of pharmaceutical compositions. Each possibility is aseparate embodiment of the invention.

According to other embodiments, the pharmaceutical composition is a longacting, controlled release, extended release or slow releaseformulation. Each possibility is a separate embodiment of the invention.

The pharmaceutical compositions of the invention are suitable for use ina variety of drug delivery systems as detailed hereinbelow.Pharmaceutically acceptable carriers suitable for use in the presentinvention may be found in Remington's Pharmaceutical Sciences, MackPublishing Company, Philadelphia, Pa., 17th ed. (1985).

According to some embodiments, the method of the invention comprises thesteps of (i) determining the risk of a subject for an autoimmunedisease; (ii) selecting a subject having a risk for said disease; and(iii) treating said subject having the risk with the PC-conjugate of theinvention.

According to some embodiments, the method of the invention comprises thesteps of (i) identifying a subject in a risk for an autoimmune disease;and (iii) treating said subject with the PC-conjugate of the invention.

Lupus, or lupus erythematosus, as used herein, refers to a category fora collection of systemic autoimmune diseases. Symptoms of these diseasesmay affect many different body systems, including joints, skin, kidneys,blood cells, heart, and lungs. Four main types of lupus are known todate: systemic lupus erythematosus, discoid lupus erythematosus,drug-induced lupus erythematosus, and neonatal lupus erythematosus. Ofthese, systemic lupus erythematosus is the most common and serious formof lupus. The abnormal immune response allows sustained production ofpathogenic autoantibodies and immune complexes that cause damage to thevarious tissues and systems. The abnormal immune response probablydepends upon the interaction of multiple hereditary and environmentalfactors.

The terms “rheumatoid arthritis” and “RA”, as used herein areinterchangeable and refer to a chronic disease featuring persistentinflammatory synovitis, typically involving peripheral joints in asymmetric distribution. This inflammation may lead to bone erosions,cartilage damage and joint destruction. It is an affliction of about 1%of the population. The prevalence increases with age, and women areaffected more frequently than men. The propagation of RA is animmunologically mediated event driven by CD4⁺ Th1 cells.

The terms “Multiple sclerosis” and “MS”, as used herein areinterchangeable and typically refer to a chronic relapsing, multifocalinflammatory disorder of the central nervous system that leads to focaldemyelination and scarring of the brain. It is a frequent diseaseaffecting about 350,000 Americans, manifesting during early to middleadulthood. MS is an autoimmune disease mediated at least in part by Th1cells. The lesions of MS resemble those induced by delayedhypersensitivity responses that contain activated T cells andmacrophages. Experimental autoimmune encephalomyelitis, also namedExperimental Allergic Encephalomyelitis (EAE) is an animal model ofbrain inflammation. It is an inflammatory demyelinating disease of thecentral nervous system (CNS). It is mostly used with rodents and iswidely studied as an animal model of the human CNS demyelinatingdiseases, including the diseases multiple sclerosis and acutedisseminated encephalomyelitis (ADEM). EAE is also the prototype forT-cell-mediated autoimmune disease in general.

The term “colitis” as used herein refers to any one or more of thefollowing diseases and disorders: inflammatory bowel disease (IBD),Crohn's disease, ulcerative colitis, collagenous colitis, lymphocyticcolitis, ischemic colitis, diversion colitis, Behcet's disease andindeterminate colitis, among others.

While the cause of IBD remains undetermined, it is presumed to resultfrom dysregulation of the intestinal mucosal immune system. Inflammatorycells in the mucosa normally have a protective effect against luminalcontents. This highly effective chronic inflammation is tightlycontrolled to limit tissue injury. IBD may result from inappropriatelyvigorous immune responses to luminal factors. Crohn's disease (CD)appears to be an overly vigorous Th-type inflammation that produces IFNγand TNFα. The incidence of Crohn's disease in industrialized societieshas increased from the 1950s until the mid-1980s, and now is about 1 to8 cases per 100,000 persons per year. This suggests that unknown changesin our environment have affected the frequency of Crohn's disease. Thenature of ulcerative colitis (UC) is less well defined.

There are several animal models of chronic intestinal inflammation. Infact, mice with genetically engineered gene deletions may developchronic bowel inflammation similar to IBD. These include mutant micebearing targeted deletions for IL-2, IL-10, and MHC class II or TCRgenes among others. It was shown in some animal models that adysregulated immune system itself can mediate intestinal injury. Themucosal inflammation in several animal models generates large amounts ofIFNγ and TNFα, suggesting that excess production of Th1-type cytokinesis one common mechanism underlying the pathogenesis of disease. Also,blocking Th1 circuitry prevents the inflammation. CD is a Th1 response.Thus, these models may have direct implications in the immunopathologyof this human disease process.

According to some embodiments, the method of the invention is fortreating colitis wherein the carrier of the PC-conjugate is a tuftsinderivative.

According to some embodiments, the method of the invention is fortreating colitis.

According to some embodiments, the disease or disorder is an autoimmuneskin disorder. There are many different types of skin-related autoimmunedisorders, including, for example scleroderma, psoriasis,dermatomyositis, epidermolysis bullosa and bullous pemphigoid. Pemphigusvulgaris is a chronic blistering skin disease with skin lesions that arerarely pruritic, but which are often painful. The disease is caused byantibodies directed against both desmoglein 1 and desmoglein 3 resultingin the loss of cohesion between keratinocytes in the epidermis. It ischaracterized by extensive flaccid blisters and mucocutaneous erosions.Psoriasis occurs when the immune system mistakes the skin cells as apathogen, and sends out faulty signals that speed up the growth cycle ofskin cells. The disorder is a chronic recurring condition that varies inseverity from minor localized patches to complete body coverage.Fingernails and toenails are frequently affected (psoriatic naildystrophy) and may be seen as an isolated symptom. Psoriasis may alsocause inflammation of the joints, which is known as psoriatic arthritis.

Thus, the invention provides compositions for parenteral administrationthat comprise a solution of the agents described above dissolved orsuspended in an acceptable carrier, such as an aqueous carrier. Avariety of pharmaceutically acceptable aqueous carriers may be used,e.g., water, buffered water, 0.4% saline, 0.3% glycine hyaluronic acidand the like. These compositions may be sterilized by conventional, wellknown sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. Each possibility is a separate embodiment of the invention.

The pharmaceutical compositions of the invention may be in the solidstate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable carriers may be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. Pharmaceutical formulations suitable for oraladministration wherein the excipient is solid are, for example,presented as unit dose formulations such as boluses, capsules or tabletseach containing a predetermined amount of the PC-conjugate of theinvention as the active compound. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules optionallymixed with a binder, lubricant, inert diluents, lubricating agent,surface-active agent or dispersing agent. Molded tablets may be made bymolding the active compound with inert liquid diluents. Tablets may beoptionally coated and, if uncoated, may optionally be scored. Capsulesmay be prepared by filling an active compound, either alone or inadmixture with one or more accessory ingredients, into the capsuleshells and then sealing them in the usual manner. Cachets are analogousto capsules wherein an active compound together with any accessoryingredient(s) is sealed in a rice paper envelope. An active compound mayalso be formulated as dispersible granules, which may, for example, besuspended in water before administration, or sprinkled on food. Thegranules may be packaged, e.g., in a sachet.

For aerosol administration, the pharmaceutical compositions of theinvention are, for example, supplied in finely divided form along with asurfactant and optionally a propellant as pharmaceutically acceptablecarriers. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Each possibility is a separateembodiment of the invention. Mixed esters, such as mixed or naturalglycerides, may be employed. A carrier may also be included, as desired,as with, e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention may be in the formliposome. Liposomes provide another delivery system for the delivery andpresentation of the immunomodulatory molecules of the invention.Liposomes are bilayer vesicles composed of phospholipids and othersterols surrounding a typically aqueous center where the PC-conjugate orother products may be encapsulated. The liposome structure is highlyversatile with many types range in nanometer to micrometer sizes, fromabout 25 nm to about 500 μm. Liposomes have been found to be effectivein delivering therapeutic agents to dermal and mucosal surfaces.Liposomes may be further modified for targeted delivery by, for example,incorporating specific antibodies into the surface membrane. The averagesurvival time or half-life of the intact liposome structure may beextended with the inclusion of certain polymers, such as polyethyleneglycol, allowing for prolonged release in vivo. Liposomes may beunilamellar or multilamellar.

The pharmaceutical compositions of the invention may be in the form ofmicroparticles and nanoparticles. Microparticles and nanoparticlesemploy small biodegradable spheres which act as depots for drugdelivery. The major advantage that polymer microspheres possess overother depot-effecting adjuvants is that they are extremely safe and havebeen approved by the Food and Drug Administration in the US for use inhuman medicine as suitable sutures and for use as a biodegradable drugdelivery. The rates of copolymer hydrolysis are very well characterized,which in turn allows for the manufacture of microparticles withsustained release of the immunomodulators over prolonged periods oftime.

The pharmaceutical compositions of the invention may be parenteraladministered as microparticles. Microparticles elicit long-lastingimmunity, especially if they incorporate prolonged releasecharacteristics. The rate of release may be modulated by the mixture ofpolymers and their relative molecular weights, which will hydrolyze overvarying periods of time. Without wishing to be bound to theory, theformulation of different sized particles (1 μm to 200 μm) may alsocontribute to long-lasting immunological responses since large particlesmust be broken down into smaller particles before being available formacrophage uptake. In this manner a single-injection vaccine could bedeveloped by integrating various particle sizes, thereby prolongingTD-PC-conjugate presentation.

The terms “vaccine composition” and “vaccine” as used herein areinterchangeable and refers to a product, the administration of which isintended to elicit an immune response that is capable of preventingand/or lessening the severity of one or more autoimmune diseases ordisorders and inflammation among other diseases and disorders.

The invention also provides a kit for the treatment of an autoimmunedisease comprising one or more containers filled with the pharmaceuticalcomposition described above. Optionally associated with suchcontainer(s) may be a notice in the form described by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

According to some embodiments, the present invention provides a kit forthe treatment of an autoimmune disease or disorder, the kit comprising afirst container comprising a phosphorylcholine-conjugate as describedabove; a second container comprising a pharmaceutically acceptablecarriers, diluents or excipients; and, optionally, a third containercomprising an adjuvant.

The TD-PC-conjugates of the invention are useful in vaccines and inimmunization protocols for prevention, treatment and progress inhibitionof autoimmune diseases, particularly inflammatory diseases.

According to some embodiments, the TD-PC-conjugates of the presentinvention, when administered to a mammal, elicit the mammal'simmunomodulation activity. A variety of models known to those skilled inthe art may be used to establish the immunomodulation ability of theconjugates of the invention. Cell cultures may be used to test theeffect of the PC-conjugates on cell proliferation, cytokine profile,development of tolerogenic dendritic cells and development of Tregulatory cell. For example, commercial kits for following pro- andanti-inflammatory cytokine, including, but not limited to IL-1, IL-2,IFNγ, IL-4, IL-10, IL-15, IL-17 and TNFα may be used. Animal models, asare known to a person skilled in the art and as exemplified hereinbelow, may be used to test the activity of the PC-conjugates of theinvention in treating, preventing or reducing the progression ofautoimmune diseases.

According to some embodiments, the immunomodulation capability of theTD-PC-conjugates of the present invention are exemplified hereinbelow inanimal models of autoimmune diseases, including collagen inducedarthritis (CIA), colitis and systemic lupus erythematosus (SLE).

The examples hereinbelow are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art may readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1: The Effect of PC-Conjugates on the Development ofSystemic Lupus Erythematosus (SLE)

Phospholipids are non-immunogenic. Therefore, phosphorylcholine (PC) iscommonly used as a conjugate to different carriers. The carriers mayserve as immunomodulatory molecules. The following PC-conjugates wereused in the current study: PC-Ovalbumin (PC-OVA; Biosearch Technologies,Inc.) and 2-methacryloyloxyethyl-PC (MPC).

In MPC, the PC appears as a moiety on the helminthes. Specifically, MPCis PC-moiety on polymer (2-methacryloyloxyethyl) core defined as MPC.MPC was purchased from NOF Inc. The potential of PC-conjugates toimmunomodulate lupus was studied in NZBxNZW/F1 mice which develop lupuson a genetic background.

Mice: female NZBxNZW/F1 mice were obtained from Harlan Olac, andsupplied by Jackson Laboratory (Bar Harbor, USA) at the age of 8-10weeks. The mice were maintained at the Sheba animal facilities, ShebaMedical Center, Israel. All animals were cared for as approved by theEthical Review Committee of the Israeli Ministry of Health and Sciencesanimal guidelines.

The serological and clinical manifestation of lupus nephritis inNZBxNZW/F1 mice was assessed as previously described (Shoenfeld Y etal., Int. Immunol. 2002, 14(11): 1303-1311). Briefly, antibodiesspecific for PC were measured by ELISA as described hereinbelow.Antibodies specific for dsDNA were detected by ELISA as previouslydescribed by us (Shoenfeld et al., 2002, ibid). Proteinuria was measuredby a standard semi-quantitative test using an Albustix kit (BayerDiagnostic). Renal immune complex deposits (ICD) were determined aspreviously described (Shoenfeld et al., 2002, ibid). The intensity ofthe ICD was graded as follows: 0, no ICD; 1, low intensity; 2, moderateintensity; and 3, high intensity of ICD. ICD analysis was performed bytwo subjects unaware whether or not each mouse is a treatment mouse or acontrol mouse.

The results demonstrate the inhibitory effect of PC-OVA and MPCtreatments on the development of lupus nephritis in the NZBxNZW/F1 lupusmice. The inhibitory effect was manifested by postponed proteinuria anddecreased immune complex deposits on the glomerular basement membrane.The results are based on treatment at an early stage of the diseasestarting at week 8 of age. PC-OVA, MPC or PBS were given 3 times a week3 μg/0.1 ml per mouse subcutaneously, n=20 per group. The mice were bledevery 2 weeks for measurement of PC and dsDNA specific antibodies.Proteinuria was assessed every 2 weeks.

Treatment with PC-OVA conjugate (FIG. 1, squares) was immunogenic andthe mice developed elevated titers of anti-PC antibodies (p<0.001) incomparison to PBS (FIG. 1, diamonds) and MPC (FIG. 1, triangles) treatedgroup, whereas MPC (poly PC on polymeric core) was non-immunogenic anddid not cause generation of anti-PC antibodies (p>0.05). Data of theanti-PC antibodies are presented at sera dilution of 1:400. The resultsare given as O.D. at 405 nm. The presence of PC specific antibodies inthe mice sera was detected by ELISA to PC-KLH (PC-Keyhole limpethemocyanin), where binding to KLH served as a control reference. Noresponse to KLH was observed. PC-OVA and MPC did not have any effect onanti-dsDNA antibodies generation in all time points (data not shown).

No effect of PC-OVA and MPC treatment was documented in anti-dsDNAantibodies generation as illustrated in FIG. 1. Proteinuria (defined asat least 100 mg/dl protein in the urine) in lupus mice treated withPC-OVA (FIG. 2, square) or MPC (FIG. 2, triangle) was postponedsignificantly (p<0.02) in comparison to treatment with PBS (FIG. 2,diamond) at 30 to 33 weeks of age.

Kidney sections from lupus mice treated with PC-OVA (FIGS. 3A-3B), MPC(FIGS. 4A-4B) and PBS (FIGS. 5A-5C) were analyzed forglomerulonephritis. Two staining procedures were applied (i)histological staining (PAS); and (ii) immunohistological staining ofimmune complex deposits in the mesangium of the kidney usinganti-mouse-Fc-FITC conjugate. As illustrated in FIGS. 5A-5C, micetreated with PBS showed severe (stage VI) glomerulonephritis exemplifiedby strong immune complex deposits (5A), diffused proliferatingglomerulonephritis (5B) and crescent necrotizing glomerulonephritis(5C). However, at the same time point of 34 weeks lupus mice treatedwith MPC (FIGS. 4A-4B) and PC-OVA (FIGS. 3A-B), exhibited only earlystage (stage II) of glomerulonephritis.

A Kaplan-Meier analysis indicates that the survival time of lupus micetreated with PC-OVA (FIG. 6, triangle) was significantly longer (FIG. 6*=p<0.01) compared to PBS treated mice (FIG. 6, diamond). The survivaltime of MPC treated mice (FIG. 6, square) showed only tendency ofsignificance (p<0.04).

Example 2: The Effect of PC-Tuftsin on Development of Proteinuria InVivo

Phospholipids are non-immunogenic, thus, PC was conjugated to tuftsin(threonine-lysine-proline-arginine, TKPR; SEQ ID NO: 17) which may serveas an immunomodulatory molecule. In autoimmune models, such as EAE andlupus, PC and PC conjugated to keyhole limpet hemocyanin (KLH) or bovineserum albumin (BSA) had an inhibitory effect on disease progression(Dagan S et al., J. Biol. Response Mod. 1987, 6(6):625-636). PCconjugated to tuftsin was synthesized based on Michaelson Chemistry. ThePC derivative, 4-aminophenylphosphorylcholine, used for the conjugateconstruction, was purchased from Biosearch Technologies.

The effect of PC-tuftsin (TPC) on proteinuria was determined in a mousemodel for lupus, specifically, female NZBXW/F1 mice that develop lupuson a genetic background. Proteinuria levels were measured usingMultistix (Bayer Diagnostics). This type of measurement is typicallyused in the clinic for assessing manifestation of lupus nephritis. Lupusnephritis is an inflammation of the kidney caused by systemic lupuserythematosus (SLE).

The mice, from age of 14 weeks, received subcutaneously PC-tuftsin atthe concentration of 50 μg/0.1 ml per mouse (treatment; n=10) or PBS(control; n=10), three times a week. Proteinuria was defined as having aprotein level of above 100 mg/dl in the urine.

The results demonstrate the inhibitory effect of PC-tuftsin treatment onthe development of glomerulonephritis. As shown in FIG. 7, a significantattenuation (p<0.02) in proteinuria was exhibited in the group of micetreated with PC-tuftsin (TPC) in comparison with the control group, i.e.mice which received only the carrier (PBS).

Example 3: The Effect of PC-Tuftsin on the Morphology of Kidneys In Vivo

The effect of PC-tuftsin on the morphology of kidneys was determined ina lupus mouse model as in Examples 2. Kidney section obtained fromkidneys of mice sacrificed after treatments were paraffin embedded. PASstaining was used to detect pathology of nephritis. Immunofluorescencestaining was used for detecting immune complex deposits. The latter wasimplemented by incubating the paraffin embedded sections withFITC-conjugated-anti-mouse-IgG. Evaluation was performed by apathologist.

Mice, from age of 14 weeks, received subcutaneously PC-tuftsin at theconcentration of 50 μg/0.1 ml per mouse (treatment; n=10) or PBS(control; n=10), three times a week.

The results demonstrate a significant inhibitory effect of PC-tuftsin onthe development of glomerulonephritis. Specifically, as illustrated inFIGS. 8A-8B, mice treated with PC-tuftsin did not display any pathologyof nephritis, a normal glomeruli is exemplified (FIG. 8B; ×60). Whereasthe control group, i.e. mice which received only the carrier (PBS),presented a severe glomerulonephritis, strong destruction of theglomeruli, as well as infiltration of lymphocytes (FIG. 8A; ×60).

As shown in FIGS. 9A-9B, in lupus mice treated with PC-tuftsin, verymild immune complex deposits were exhibited in the glomeruli (FIG. 9B;×40) as compared to the control group (PBS), which present severe immunecomplex deposits (FIG. 9A; ×20).

Example 4: The Effect of PC-Tuftsin on the Immune System In Vivo

The effect of PC-tuftsin on the immune system was determined in thelupus mouse model as in Examples 2. Cytokines analyses and T regulatorycell profiling were measured; these types of studies are typically usedin the clinic for assessing manifestation of lupus nephritis.

Mice, from age of 14 weeks, received subcutaneously PC-tuftsin at theconcentration of 50 μg/0.1 ml per mouse (treatment; n=10) or PBS(control; n=10), three times a week. In the cytokines studies, therelative mRNA expression levels of pro-inflammatory cytokine IFNγ andanti-inflammatory cytokine TGFβ were analyzed by real time RT-PCR usingLightCycler (Roche). Total RNA was isolated from splenocytes and wasreverse-transcribed into cDNA by using Moloney Murine Leukemia VirusReverse Transcriptase (Promega). The resulting cDNA was subjected toreal-time RT-PCR in the presence of specific primers, according to themanufacturer's instructions (Table 2). The relative expression levels ofIFNγ and TGFβ were normalized to β-actin levels.

TABLE 2 RT-PCR primers (forward and reverse respectively) SEQ. PrimerSequence (5′ to 3′) ID NO: IFNγ gaacgctacacactgc  1 IFNγctggacctgtgggttg  2 IL-1β ccccaactggtaaatca  3 IL-1β ccgaggactaaggagtg 4 IL-10 aacctcgtttgtacctct  5 IL-10 caccatagcaaagggc  6 IL-17agggcaagggatgctctctag  7 IL-17a ctgaagctgctgcagagctg  8 TNF-αacgtcgtagcaaaccac  9 TNF-α agatagcaaatcggctg 10 TGF-β gaacccccattgctgt11 TGF-β gccctgtattccgtct 12 Foxp3 taccacaatatgcgaccc 13 Foxp3ctcaaattcatctacggtcc 14 β-actin gtgacgttgacatccg 15 β-actincagtaacagtccgcct 16

The results demonstrate that PC-tuftsin (TPC) exerts a significantinhibitory effect on the development of glomerulonephritis. As shown inFIG. 10, the splenocytes relative mRNA expression of anti-inflammatorycytokine TGFβ in mice treated with PC-tuftsin was significantly enhancedin comparison to the TGFβ mRNA derived from the PBS treated mice(p<0.001). In contrast, the splenocytes relative mRNA expression levelof pro-inflammatory cytokine IFNγ in mice treated with PC-tuftsin (TPC)was significantly reduced, i.e. ameliorated in comparison to the IFNγmRNA of the PBS control group (p<0.03). The results indicate thatPC-tuftsin inhibited development of inflammation associated withnephritis.

Splenocytes protein level of anti-inflammatory cytokines TGFβ, and IL-10and pro-inflammatory cytokines IFNγ, and IL-17 were quantified by DuoSet(R&D Systems), according to the manufacturer's instructions. Briefly,spleens from mice treated with PC-tuftsin or PBS were harvested,splenocytes were isolated (5×10⁶/ml) and incubated for 72 hours andthereafter assessed for contents of secreted cytokines.

The results demonstrate a significant inhibitory effect exerted byPC-tuftsin on the development of glomerulonephritis. As shown in FIG.11, mice treated with PC-tuftsin (TPC) exhibited 5.1 and 4.8 increase inthe protein levels of anti-inflammatory cytokines, TGFβ and IL-10,respectively, in comparison to PBS treatment (p<0.001). At the sametime, the concentrations of pro-inflammatory cytokines IFNγ and IL-17 inPC-tuftsin (TPC) treated mice reduced by 5.2 and 2.7 fold, respectivelyas compared to control group (p<0.001, p<0.02, respectively). Theseresults further indicate that PC-tuftsin treatment results with asignificant inhibition of pro-inflammatory cytokines and enhancedexpression of anti-inflammatory cytokines, thereby reduces, and evenprevents, the occurrence of nephritis.

Finally, a T regulatory cell (Tregs) profiling assay was performed inisolated splenocytes incubated with the following antibodies:anti-CD4⁺FITC, anti-CD25⁺APC and anti-FOXP3⁺PE (eBioscience) andanalyzed by FACS. Forward and side scatter gates adjusted to include allcells and to exclude debris (Becton Dickinson). Cells were gated on CD4+cells, and for intracellular staining, the cells were incubated with afixation solution, washed and resuspended in permeabilization solution(Serotec). Isotype control was used as reference.

Here too, the results demonstrate a significant inhibitory effect ofPC-tuftsin treatment on the development of glomerulonephritis. As shownin FIG. 12 mice treated with PC-tuftsin (TPC) presented an enhancementof 18±2 percent in the Tregs, CD4⁺CD25⁺FOXP3⁺ expression level (p<0.02),whereas with PBS treated mice an increase of only 4±0.6 percent wasobserved (p<0.01). Notably, phosphorylcholine (PC) or tuftsin (T) didnot cause any significant elevation in Tregs level, (p<0.01). Moreover,the relative amount of Tregs obtained with PC-tuftsin is higher than thesum of relative Tregs obtained from each of PC and tuftsin. Thus, thePC-tuftsin exhibits a synergistic therapeutic effect.

Example 5: Synthesis of a Tuftsin-Derived-Phosphorylcholine (TD-PC)Conjugate

Tuftsin was extended at its C-terminal thereby formingThr-Lys-Pro-Arg-Gly-Tyr (TD; SEQ ID NO: 25). TD-PC was synthesizedmanually following solid phase peptide technology. The peptide wascoupled to diazotized 4-aminophenyphosphorylchloride to form an azo bondbetween the tuftsin derivative (SEQ ID NO: 25) and PC. The conjugateTD-PC was characterized by mass spectra, amino acid analysis as well asby High-performance liquid chromatography (HPLC).

Example 6: The Effect of PC-Tuftsin Derivative on Colitis Development InVivo

The effect of PC-tuftsin derivative (SEQ ID NO: 25) on colitisdevelopment was determined in a mouse model subjected to induction ofacute colitis by dextran sulfate sodium (DSS) treatment. Specifically,induction of colitis was carried out in male C57BL/6 mice (“colitismice”) by supplementing the drinking water (2.5% wt/v) for five dayswith DSS (mol. wt. 36,000-50,000).

Two groups of mice were fed daily via oral ingestion using afeeding-needle, with PC-tuftsin derivative at a concentration of 500μg/0.1 ml per mouse (treatment; n=10), or PBS (control; n=10). Thesecompounds were given during 11 days starting two days before DSSadministration. A third group of mice (n=10) were not induced withcolitis, i.e. did not receive DSS, nor any treatment. All the micegroups had an identical average body weight (27-29 gr).

Assessment of colitis development was performed by monitoring bodyweight, rectal bleeding, stool consistency, and survival every secondday. Intestinal bleeding was followed by Hemoccult test and observationof bleeding signs on the anus or gross bleeding. The daily diseaseactivity index (DAI) was calculated by grading on a scale of zero tofour the following parameters: change in weight (0, <1%; 1, 1-5%; 2,5-10%; 3, 10-15%; and 4, >15%), intestinal bleeding (0, negative; 4,positive), and stool consistency (0, normal; 2, loose stools; 4,diarrhea). The combined scores were then divided by three to obtain thefinal disease activity index. Ten days following disease induction, micewere sacrificed and the large intestine was collected and evaluated forcolon length and microscopic colonic damage. For microscopic scoring,the proximal, medial, and distal portions of the colon and the cecumwere fixed in 10% phosphate-buffered formalin. Paraffin-embeddedsections were stained with H&E. The degree of histological damage andinflammation was graded in a blinded fashion by an expert pathologist

The results demonstrate an amelioration effect of PC-tuftsin derivativeon the development of DSS induced colitis. As shown in FIG. 13, the DAIscore of colitis mice treated with PC-tuftsin derivative (empty circle)was around 0.9 (p<0.02) at the last day of the experiment, day 8, whichwas significantly lower than the DAI of the control group (PBS, solidcircle). The latter gradually increased with time reaching the value of2.6 after 5 days.

Furthermore, as illustrated in FIGS. 14-15, mice treated with PC-tuftsinderivative (empty circle) exhibited significantly low rectal bleeding(FIG. 14; p<0.001) and loss of weight (FIG. 15; p<0.001) in comparisonwith the control (PBS treated, solid circle) mice. Finally, asillustrated in FIG. 16, mice treated with PC-tuftsin derivative (emptycircle) exhibited significantly higher survival. After 5 days, a drop inthe survival was exhibited with the PBS treated mice (solid circles),whereas 100% of the mice treated with PC-tuftsin (empty circle) werestill alive, even after day 12.

As exemplified in FIGS. 17-18, the colon length of mice treated withPC-tuftsin (TD-PC) was eight cm, in comparison with the control (PBStreated) mice, which shortened from eight cm to five-six cm (p<0.02).

Finally, as shown in FIG. 19, panels A-F, histological analyses of colonsection from the colons of all three mice groups revealed thatPC-tuftsin derivative (TD-PC) treatment (FIG. 19—panel B ×20, panel E×60) attenuated colon destruction as oppose to the control treatments,namely, PBS treatment (FIG. 19—panel A ×10, panel D ×40) and healthymice, i.e. mice not subjected to DSS induction of colitis (FIG. 19—panelC ×20, panel G ×60). No difference was noted in the structure of thecolon epithelia in healthy mice (FIG. 19, panel C, panel F) nor incolitis mice treated with TD-PC (FIG. 19, panel B, panel E), whereas astrong infiltration of cells and zoom out of the glands were seen incolitis mice treated with PBS (FIG. 19, panel A, panel D).

Example 7: Cytokines Expression in the Colon

The expression of the proinflammatory cytokines (TNFα, IL-17, IL-β) andanti-inflammatory cytokine IL-10 was analyzed by immunoblot in colonlysates. The expression of TNFα, IL-17 and IL-β was suppressed in celllysates taken from TD-PC-treated mice, suggesting that TD-PC (SEQ ID NO:25 conjugated to PC) attenuated the expression of the pro-inflammatorycytokines. Significant increased IL-10 expression was documented,suggesting that TD-PC enhanced anti-inflammatory cytokine expression inthe colon (FIG. 20).

Example 8: The Effect of TD-PC on Proteinuria Onset

To examine the potential protective effect of TD-PC (SEQ ID NO: 25conjugated to PC), female NZBxW/F1 lupus prone mice receivedsubcutaneous injections (s.c) of the TD-PC conjugate, phosphorylcholine(PC), tuftsin derivative (SEQ ID NO: 25) or PBS (control) via aprophylactic protocol starting at week 14, before the clinical onset oflupus (n ¼ 15 per group). As illustrated in FIG. 21, at the age of 30weeks, 72% of the mice treated with PBS developed proteinuria whereasonly 21% of the TD-PC treatment group had proteinuria above 100 mg/dl.The TD-PC-treated mice showed a significant reduced onset of proteinuriaas compared with control mice that received PBS, tuftsin derivative (SEQID NO: 25) or PC, all which when given separately served as controls forTD-PC (p<0.001, p<0.02, p<0.03 respectively). Without being bound by anytheory or mechanism, it is concluded that TD-PC reduced the appearanceof immune complex (IC) deposits in the mesangium of the TD-PC treatedmice to class-II nephritis. In contrast, treatment withphosphorylcholine or PBS did not diminish the development of strongmesangial damage defined as class-IV. Tuftsin derivative (SEQ ID NO: 25)alone had a moderate but not significant effect on IC deposition.

Example 9: TD-PC Prolong of the Survival Time of Lupus Mice

A main complication of lupus nephritis is a short survival time due tosevere kidney failure. Therefore, the survival time of mice treatedeither with TD-PC or with PC, tuftsin derivative (SEQ ID NO: 25) or PBS(control) was analyzed. As depicted in FIG. 22, a significant differencein survival time between the TD-PC treated mice and the control groups,between weeks 24th up to 40th week was observed. Additionally, asignificant difference in the percentage of death number in mice wasdocumented between PBS injected mice in comparison to PC, tuftsinderivative (SEQ ID NO: 25) and TD-PC treated mice already at week 24.

Example 10: TD-PC Therapy and Titers of Circulating Anti-dsDNAAntibodies

Titers of circulating anti-dsDNA antibodies in the sera of control andtreated mice were monitored during the development of lupus, and theirlevels were measured every 3 weeks. No statistically significant changewas noticed between the group of mice treated with TD-PC (SEQ ID NO: 25conjugated to PC), tuftsin derivative (TD; SEQ ID NO: 25), PC or PBS(p>0.05, FIG. 23). FIG. 23 presents the anti dsDNA antibodies titers inthe sera at the age 30 weeks at a dilution of 1:800. A follow-up ofanti-dsDNA antibodies over time (16-40 weeks) did not show anysignificant change between the different groups of mice (p>0.05; datanot shown).

Example 11: Cytokines Profile under Treatment of Lupus with TD-PC

Cytokines were shown to play a major role in the pathogenesis of lupus.NZBXW/F1 female mice having lupus were treated with TD-PC (SEQ ID NO: 25conjugated to PC) or the corresponding control treatments. Mice weresacrificed at week 30, and their splenocytes were studied in-vitro forthe expression of pro-inflammatory cytokines (IFNγ, IL-17) andanti-inflammatory (TGFβ, IL-10) cytokines using RT-PCR. It was foundthat TD-PC inhibited the mRNA expression of the pro-inflammatorycytokine IFNγ in comparison to the mRNA expressed in PBS treated mice(p<0.03; FIG. 24A). In contrast, TD-PC increased the mRNA expression ofthe anti-inflammatory cytokine TGFβ in the (p<0.001) compared to themRNA expressed by splenocytes derived from PBS-treated mice (FIG. 24A).

Striking inhibition of pro-inflammatory cytokines (IFNβ, IL-17)secretion in-vitro by spleen cells originated from TD-PC treated mice isdemonstrated in FIG. 24B. However, a prominent increase inanti-inflammatory cytokines (TGFβ, IL-10) secretion in-vitro bysplenocytes derived from TD-PC treated mice is illustrated in FIG. 24B.TD-PC treatment reduced splenocyte secretion of IFNγ from lupus TD-PCtreated mice by 5.2 times, compared to IFNγ secretion in spleen cellsfrom PBS subjected mice (p<0.008; FIG. 24B). IFNγ secretion upon tuftsinderivative (SEQ ID NO: 25) or PC treatments was reduced by 2.5 and 1.5times respectively (p<0.03 and p<0.04, respectively). A significantdecline was observed in the levels of IL-17 secretion from splenocytes,in vitro, from TD-PC treated mice, (p<0.01), compared to control (PBS)mice. Less significant inhibition of IL-17 secretion was induced bytuftsin derivative (SEQ ID NO: 25; p<0.03) and to a lower extent by PC(p<0.04). TD-PC significantly upregulated the production of theanti-inflammatory cytokine TGFβ splenocytes in-vitro (p<0.007), whencompared to splenocyte secretion in control (PBS) mice. Tuftsinderivative (SEQ ID NO: 25) moderately enhanced the secretion of TGFβ,while PC had a minimal effect on this cytokine expression, (p<0.05).TD-PC remarkably increased IL-10 concentration in the culture fluid ofsplenocytes derived from TD-PC treated mice in comparison to splenocytesfrom control (PBS) mice (p<0.001). Treatment with tuftsin derivative(SEQ ID NO: 25) also increased IL-10 secretion by splenocytes in-vitrobut less significant than treatment with TD-PC (p<0.01). Splenocytesfrom mice treated with PC did not have any effect on IL-10 secretionin-vitro.

Example 12: The Effect of TD-PC on the Expansion of Treg Cells

The percentage of CD4+CD25+FOXP3+ Tregs phenotype subset in spleen cellsfollowing treatment with TD-PC, tuftsin derivative (SEQ ID NO: 25), PCor PBS, is elucidated in FIGS. 25A-25B. A marked increase in Tregsphenotype was observed in the TD-PC treated group of mice when comparedto Tregs levels in spleen cells of mice treated with tuftsin derivative(SEQ ID NO: 25), PC or PBS (18%, 8%, 3% and 4% respectively).Representative data of FACS scans gating on CD4+ T cells are presentedin FIG. 25A. Statistics of several FACS analyses (N=15) is provided inFIG. 25B. The results indicate that TD-PC promoted significantly theTregs phenotype expansion (p<0.03) compared to treatments/controls.Treatment with tuftsin derivative (SEQ ID NO: 25) and PC did not lead toTregs expansion (p>0.05).

Example 13: TD-PC Reduces Arthritis in Mice with CIA

The main characteristic of rheumatoid arthritis is joint deformation dueto high inflammation. The TD-PC effect on arthritis score in CIA micewas monitored. CIA mice were treated with TD-PC, orally or s.c. Controlmice were treated with PBS orally or s.c. As detailed in FIGS. 26A and26B, significantly lower arthritis score was observed in TD-PC treatedmice compared to control mice (p<0.05). Arthritis score was lower inboth groups of TD-PC treatment (oral and s.c.) starting two weeks afterdisease induction—day 14, until the mice were sacrificed —day 31. At day31, TD-PC treated groups had a mean arthritis score of 1.5 (ranged from0 to 4), while the control groups had mean arthritis score of 11.8(ranged from 10 to 14; P<0.001). TD-PC s.c treated mice had a meanarthritis score of 1.4±0.84 whereas, PBS s.c treated mice had a meanarthritis score of 11±1.22 (p<0.001). Moreover, TD-PC oral treated micehad a mean arthritis score of 1.6±1.5 while, PBS oral treated mice hadmean arthritis score of 12.6±1.14 (p<0.001). Representative pictures ofmice joints are shown in FIG. 26C, demonstrating significant differencesin inflammation between the TD-PC treated mice (lower panels C and D) incomparison to the control mice (upper panels A and B). Control micedeveloped edema and erythema at the ankle and towards the entire legwhile TD-PC treated mice exhibited milder symptoms.

Example 14: TD-PC Effect on Titers of Anti-Collagen Antibodies in MiceSera

Titers of anti-collagen type II antibodies in mice sera at a dilution of1:200 (OD at 405 nm) were measured at day 30 after disease inductionwhile arthritis was fully manifested (FIG. 27). A statisticallysignificant difference was documented between the TD-PC s.c and oraltreated mice in comparison to PBS s.c and oral treated mice (p<0.001).The mean OD level of the anti-collagen type II antibodies in the micetreated with TD-PC per os was 0.685±0.09 while the corresponding mean ODlevel in control mice treated with PBS per os was 1.09±0.17.Furthermore, the mean OD level of the anti-collagen type II antibodiesin mice treated with TD-PC s.c was 0.615±0.08, whereas the correspondingmean OD level mice treated with PBS s.c was 1.03±0.13 (p<0.001).

Example 15: TD-PC Immunomodulation of Cytokines Expression

Cytokines play a major role in synovial inflammation while the majorones are IL-17, TNFα and IL-1. Secretion of pro-inflammatory (TNFα,IL-1β, IL-17) and anti-inflammatory cytokines (IL-10) by splenocytesderived from CIA mice treated with TD-PC or PBS (control), in-vitro, wasevaluated. As depicted in FIGS. 28A-C and presented in Table 3, thelevel of pro-inflammatory cytokines in TD-PC treated mice (oral and s.c)was significantly lower compared to control mice (p<0.0001).

Furthermore, TD-PC increased the level of anti-inflammatory cytokineIL-10 in a significant manner compared to control mice (p<0.0001; FIG.28D and Table 3).

TABLE 3 TD-PC (pg/ml) PBS (pg/ml) Cytokine oral s.c. oral s.c. TNFα 633621 1677 1586 IL-17 505 497 1585 1395 IL-1β 183 198 686 770 IL-10 36052917 384 302

Example 16: The Effect of TD-PC on the Expansion of Treg Cells

The frequency of CD4+CD25+FOXP3+ Treg cells subset in isolated micesplenocytes, following treatment with TD-PC or control (PBS) iselucidated in FIGS. 29A-29B. A striking increase of Treg cells wasobserved in isolated splenocytes derived from TD-PC treated micecompared to control indicating that TD-PC treatment significantlypromoted the Treg phenotype expansion (p<0.001). Representative data ofthe CD4+CD25+FOXP3+ levels in splenocytes derived from TD-PC and PBStreated mice gating on CD4+ T cells are presented in FIG. 29A and thecorresponding statistics is presented in FIG. 29B.

Example 17: The Effect of TD-PC on Breg Cells Expansion in Splenocytes

The mean percentage of Breg cells in isolated splenocytes followingtreatment with TD-PC or PBS was measured within two main subsetsCD19+IL-10+TIM-1+ (representative data: FIG. 30A, statistics: FIG. 30C,n=15) and IL-10^(high)CD5^(high)CD1d^(high) (representative data: FIG.30B, statistics: FIG. 30D, n=15). As depicted in FIGS. 30A-D, bothsubsets demonstrated that TD-PC significantly stimulated the Bregphenotype expansion in isolated mice splenocytes (p<0.0001) compared tocontrol (PBS treated) mice.

Example 18: TD-PC Stopped Arthritis Progression in Mice with CIA

Rheumatoid Arthritis is a progressive joint deformation disease causedby severe inflammation. CIA mice were subjected to subcutaneousinjections of TD-PC, tuftsin derivative (SEQ ID NO: 25), PC, a mix of TDand PC, or PBS, 3 times a week. Untreated mice (None) did not receiveany treatment. Treatment started as day 16 after disease induction whendisease symptoms were already notable. As shown in FIG. 31A, at time 0(treatment initiation) average arthritis score was relatively equalbetween all groups (TD-PC (SEQ ID NO: 25 conjugated to PC)—3.6±0.9;PBS—3.8±0.8; TD (SEQ ID NO: 25)—4.6±1.5; PC—3.4±1.1; TD+PC mix—4±0;None—2.75±1.5). A significant lower arthritis score was observed inTD-PC treated mice compared to mice treated with PBS, TD and PC anduntreated mice (p<0.0001) or compared to TD+PC treated mice (p<0.05).The significant reduction in arthritis score was illustrious from day 7after treatment administration and lasted until day 35 (the day micewere sacrificed). At day 35, the TD-PC treated mice had a mean arthritisscore of 6.8±0.8 while control (PBS oral) mice had mean arthritis scoreof 13.8±0.45 (p<0.0001). Moreover, mice treated with the tuftsinderivative (SEQ ID NO: 25) had a mean arthritis score of 13.8±0.45, PCtreated mice had a mean arthritis score of 14±0 and untreated mice had amean arthritis score of 13±0.8 (p<0.0001). Furthermore, mice treatedwith a mix of TD+PC had a mean arthritis score of 9±2.2 (p<0.05).Representative pictures of mice joints are shown in FIG. 31B,demonstrating the less visible inflammation in the TD-PC treated mice incomparison with the PBS, TD, PC and TD+PC treated mice. TD-PC treatedmice exhibited milder arthritis symptoms compared to other groups ofmice.

Example 19: TD-PC Immunomodulation of Cytokines Expression.

Secretion, in-vitro, of pro-inflammatory and anti-inflammatory cytokinesby splenocytes derived from CIA mice treated with TD-PC, PBS, TD, PC,TD+PC or untreated mice was evaluated (FIGS. 32A-32D). The followingcytokines secretions were examined: pro-inflammatory IL-1β (FIG. 32A),IL-17 (FIG. 32B), IL-6 (FIG. 32C), TNF-α (FIG. 32D) andanti-inflammatory IL-10 (FIG. 32E). The results indicate that TD-PCtreatment inhibited the production of pro-inflammatory cytokines incomparison to controls (PBS, TD (SEQ ID NO: 25), PC and TD+PC treatmentsor no treatment; p<0.0001).

Mice treated with TD-PC had mean IL-1β level of 320 (pg/ml), mean IL-17level of 1770 (pg/ml), mean IL-6 level of 480 (pg/ml) and mean TNF-αlevel of 342 (pg/ml). While, mice treated with PBS had mean IL-1β levelof 883 (pg/ml), mean IL-17 level of 2170 (pg/ml), mean IL-6 level of 840(pg/ml) and mean TNF-α level of 1073 (pg/ml). Moreover, TD (SEQ ID NO:25) treated mice had mean IL-1β level of 896 (pg/ml), mean IL-17 levelof 2217 (pg/ml), mean IL-6 level of 778 (pg/ml) and mean TNF-α level of1079 (pg/ml). PC treated mice had mean IL-1β level of 860 (pg/ml), meanIL-17 level of 2179 (pg/ml), mean IL-6 level of 795 (pg/ml) and meanTNF-α level of 1048 (pg/ml). TD+PC treated mice had mean IL-1β level of877 (pg/ml), mean IL-17 level of 2304 (pg/ml), mean IL-6 level of 717(pg/ml) and mean TNF-α level of 1039 (pg/ml). Untreated mice had meanIL-1β level of 785 (pg/ml), mean IL-17 level of 2352 (pg/ml), mean IL-6level of 860 (pg/ml) and mean TNF-α level of 1055 (pg/ml).

TD-PC significantly increased the level of anti-inflammatory cytokineIL-10 in comparison to control (PBS) mice (p<0.001; FIG. 32E).

Mice treated with TD-PC had mean IL-10 level of 1745 (pg/ml) where, micetreated with PBS had mean IL-10 level of 298 (pg/ml). Furthermore, TDtreated mice had mean IL-10 level of 304 (pg/ml). PC treated mice hadmean IL-10 level of 300 (pg/ml). TD+PC treated mice had mean IL-10 levelof 429 (pg/ml) and untreated mice had mean IL-10 level of 363 (pg/ml).

Example 20: The Effect of TD-PC on Expansion of Treg Cells inSplenocytes

The frequency of CD4+CD25+FOXP3+ Treg cells subset in isolatedsplenocytes from mice treated with TPC, PBS, TD (SEQ ID NO: 25), PC,TD+PC or untreated is elucidated in FIGS. 33A and 33B. A remarkableincrease was observed in the mean percentage of the Treg cells measuredin isolated splenocytes derived from TD-PC treated mice (8.75%) comparedto the various controls (PBS, TD, PC and TD+PC treated mice anduntreated mice—1.4%, 0.52%, 0.81%, 0.97% and 1.33%, respectfully). Asillustrated in FIG. 33A, TD-PC treatment significantly promoted the Tregphenotype expansion (p<0.001) when compared with other treated groups ofmice. Representative data of the CD4+CD25+FOXP3+ levels in splenocytesderived from TD-PC, PBS, TD (SEQ ID NO: 25), PC and TD+PC treated miceand the untreated group, gating on CD4+ T cells are presented in FIG.33B.

Example 21: The Effect of TD-PC on Expansion of Breg Cells inSplenocytes

The mean percentage of Breg cells in isolated splenocytes followingtreatment with TD-PC, PBS, TD (SEQ ID NO: 25), PC, TD+PC and None wasmeasured within IL-10+CD25+CD1d+ subset. As depicted in FIG. 34A-34B,TD-PC significantly stimulated the Breg phenotype expansion in isolatedmice splenocytes (p<0.001) with respect to PBS, TD, PC, TD+PC andtreated groups of mice. As demonstrated in FIG. 34B, the mean percentageof IL-10+CD25+CD1d+ Breg cells of TD-PC treated mice was higher 13.73%while PBS, TD (SEQ ID NO: 25), PC and TD+PC treated groups had meanpercentage of 0.4%, 0.5%, 0.44% and 0.48% respectfully. Representativedata of IL-10+CD25+CD1d+ levels in splenocytes derived from TD-PC, PBS,TD (SEQ ID NO: 25), PC and TD+PC treated mice gating on IL-10+ B cells,are presented in FIG. 34A.

Example 22: TD-PC Induces Macrophages to Secret Anti-InflammatoryCytokines

The relative expression of the anti-inflammatory cytokine IL-10 wasanalyzed in human macrophages treated with TD-PC (TD-PC), andPC-Ovalbumin (OVA-PC). Specifically, THP-1 cells (a monocytic cell lineestablished from human monocytic leukemia) were induced todifferentiation into adherent macrophage-like phenotype (M0) byculturing the cells (10⁵ cells/ml) in the presence of phorbol12-myristate 13-acetate (PMA; 80 nM) and VD3 (100 nM). For inducingdifferentiation from M0 to M1 (M1 polarized macrophages) the cells werefurther incubated with LPS (1 μg/ml), IFNγ (20 ng/ml). For shifting fromM1 to M2 macrophages (M2 polarized macrophages) the cells were washedand further incubated with IL-4 (10 ng/ml) and IL-13 (10 ng/ml) for 72hours.

In order to determine whether TD-PC, TD, PC or PC-OVA can induce a shiftform M1 cells to M2 macrophages secreting anti-inflammatory cytokineIL-10, M1 polarized macrophages were incubated with each molecule orconjugate at 5 μg/ml for 72 hrs. As shown in FIG. 35, TD-PC induce asignificant shift of M1 macrophages to M2 macrophage in comparison toOVA-PC (p<0.00001). The results suggest that TD-PC is advantageous overOVA-PC in inducing a shift from pro-inflammatory macrophages tomacrophages secreting anti-inflammatory cytokine IL-10.

Example 23: TD-PC Shifts the Microbiome Profile

The effect of TD-PC on the microbiome of mice with collagen inducedarthritis (CIA) was monitored. A comparison of the microbiome populationbetween day 0 (before disease induction) and day 35—when the disease isestablished and the mice are inflamed was performed using PrincipalCoordinate Analyses (PCA) plot (FIGS. 36A-36B), which represent thediversity of the different bacteria populations within the fecal samplesof each treatment: (group 1) s.c. injection of the TD-PC set forth inSEQ ID NO: 25 to healthy mice (gray triangle); (group 2) s.c. injectionof the TD-PC set forth in SEQ ID NO: 25 to CIA mice (gray square);(group 3) s.c. injection of PBS to healthy mice (black circle); and(group 4) s.c. injection of PBS to CIA mice (black square). PC1 and PC2and PC3 along the x and y and z axes, respectively, depict the amount ofvariance (the spread of the data values) in the samples explained bythese components, included in brackets. The number of principlecomponents (axes) is less than or equal to the number of individualmeasurements included for each sample, with each axis describing afraction of the sample variance. As shown in FIG. 36A, a variety ofbacteria is randomly distributed at day 0. However, following treatmentwith the conjugate TD-PC, the microbiome profile associated withuntreated mice bearing the disease (group 2) is significantly distinctfrom the corresponding microbiome profiles in the remaining groups(groups 1, 3 and 4).

The results clearly indicate that microbiome profile of mice treatedwith TD-PC is similar to the microbiome profile of the healthy mice(FIG. 36B). Accordingly, the results suggest that the therapeutic effectexerted by TD-PC in CIA involves, inter alia, the ability of theconjugate to shift the microbiome of mice having CIA to a microbiome ofhealthy mice.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1. A method for treating an autoimmune disease in a subject in needthereof comprising administering to the subject a composition comprisinga phosphorylcholine-conjugate comprising at least one phosphorylcholinemoiety or a derivative thereof comprising phosphorylcholine, and atleast one carrier, said carrier comprising tuftsin.
 2. The method ofclaim 1, wherein said tuftsin comprises the amino-acid sequence TKPR(SEQ ID NO: 17).
 3. The method of claim 1, wherein saidphosphorylcholine moiety or derivative thereof comprisingphosphorylcholine is covalently bound to said at least one carriercomprising tuftsin.
 4. The method of claim 1, wherein saidphosphorylcholine moiety or derivative thereof comprisingphosphorylcholine and said at least one carrier comprising turfsin areseparated by a spacer.
 5. The method of claim 1, wherein said autoimmunedisease is associated with abnormal inflammation.
 6. The method of claim1, wherein the autoimmune disease is selected from the group consistingof rheumatoid arthritis, lupus, multiple sclerosis, pemphigus vulgaris,antiphospholipid syndrome, psoriasis, autoimmune hepatitis, sarcoidosis,inflammatory bowel disease, colitis, Crohn's disease and chronicobstructive pulmonary disease.
 7. The method of claim 1, wherein theautoimmune disease is selected from the group consisting of arthritis,lupus, and colitis.
 8. The method of claim 7, wherein the autoimmunedisease is lupus.
 9. The method of claim 7, wherein the autoimmunedisease is arthritis.
 10. The method of claim 9, wherein said arthritisis rheumatoid arthritis.
 11. The method of claim 7, wherein theautoimmune disease is colitis.
 12. The method of claim 11, wherein saidcolitis is inflammatory bowel disease (IBD) or Crohn's disease.
 13. Themethod of claim 1, wherein said treating comprises at least one ofpreventing the onset of said autoimmune disease, attenuating theprogress of said autoimmune disease and inhibiting the progression ofsaid autoimmune disease.
 14. The method of claim 1, wherein saidtreating comprises increasing T regulatory cells (Tregs) in saidsubject.
 15. The method of claim 1, wherein said treating comprisesdecreasing proteinuria in said subject.
 16. The method of claim 1,wherein said treating comprises decreasing glomerulonephritis in saidsubject.
 17. The method of claim 1, wherein said treating comprisesdecreasing inflammation in said subject.
 18. The method of claim 15,wherein decreasing inflammation comprises decreasing expression of atleast one pro-inflammatory cytokines, increasing expression of at leastone anti-inflammatory cytokines or both.
 19. The method of claim 18,wherein said at least one pro-inflammatory cytokine is selected from thegroup consisting of: interferon gamma (IFNγ) and interleukin 17 (IL-17).20. The method of claim 18, wherein said at least one anti-inflammatorycytokine is selected from the group consisting of: transforming growthfactor beta (TGFβ) and interleukin 10 (IL-10).